The invention relates to an arrangement (a measurement device) for on-line measurements on cells, in particular for measuring soluble analytes and dissolved gases on samples in a sample area.
The behavior of cell cultures, particularly with regard to the metabolic-physiological parameters, can be detected by using very different electro- or biochemical sensors.
DE 41 15 792 describes for example a principal arrangement for a biosensor system that is suitable for biochemical measuring methods by using a membrane-covered miniature electrode and is equipped with a membrane-clamping and membrane-replacing unit.
According to this publication, the counter electrode of the miniature electrode is shaped like a spherical cap that is provided with a center aperture in which the head of the meter electrode is arranged.
The membrane-clamping and membrane-replacing unit consists of a hinged lower part for changing the membrane and a second lower part, which is connected to the first lower part, can be let down and in the first mentioned lower part the membrane is pre-clamped and sealed by a Teflon ring in a cylindrical recess that is expanding a through hole.
The membrane is clamped again by the head of the miniature electrode via the center hole in the Teflon ring and sealed again by the upper edge of the isolation body.
However, this basic arrangement cannot be used for on-line measurements on cell cultures over longer periods of time because principally the membrane cannot be integrated in the sample area and is not designed for the culture of cells. Particularly, adherent cell cultures require a stable, immobile substrate. In the described arrangement, an interference-free separation of the culture space and the sensors is not possible.
In fact, it cannot be avoided that the electrochemical sensors used for the detection of the behavior of cell cultures can only record correct measured values over a limited period.
It is a generally known fact that almost all chemosensors and all biosensors, for which chemosensors mostly form the basic sensor, require a constant recalibration because for functional reasons relatively thin layers or layer systems of different compositions, organic and/or inorganic crystalline or amorphous membranes or possibly fluid functional elements are, among others, components of the sensor. Depending on several parameters (age, environment, pressure, temperature, etc.) they are subject to a change in the material that directly affects the generated sensor signal.
Thus, the arrays occupied by the cells of a cell culture can normally be used in experiments for a maximum period of only three days. That is that the usability for the online measurement is restricted to processes that can be performed within this period completely or at least to such a point at which a significant statement can be made about the course of the metabolism in question.
A lot of the suited sensor materials are not only disturbed by a coating of cells and proteins, but also their biocompatibility can be impaired. Thus, particularly adherent cells that show a sensitive reaction to the nature of the substrate cannot be analyzed in their natural condition.
For the cultivation of cells in general and for the three-dimensional, multi-layer tissue engineering implants in particular (e.g. non-vascular artificial cartilages) the control of the nutrients absorbed by the metabolism of the cells is essential for the successful cultivation during longer periods. For this it is advantageous to measure at least the physiologically important parameters oxygen, glucose and pH-value in the lowest cell layers.
So far, the determination of these parameters has been accompanied by the destruction of the samples or said parameters have been tested indirectly by measuring the culture medium. Fluorescence-based systems with fluorophores embedded in silicone are recently available but adherent cells do not grow on them. The use of the above described sensor-array systems has to be ruled out because of the impossibility of recalibration during the cultivation process.
Therefore, one of the most serious problems in the use of electrochemical sensors is the interaction of the sensor surface with the sample. Substances, such as organic molecules or proteins, can interact with the sensor and thus reduce the sensitivity of the sensor. Moreover, problems can arise when measuring live systems that can be negatively influenced by the sensor surface, e.g. by silver/silver chloride reference electrodes.
According to the prior art, fluorescence-based systems are known that are mostly bioinert and thus principally suited for the measurement on suspension cell cultures but only few adherent cell types, if at all, can grow on these systems. Such a system is, for example, disclosed in DE 199 03 506, DE 100 03 673 and WO 03/036293.
A further disadvantage of such fluorescence-based systems is the low number of fluorophore systems that are available at present.
Sensor systems that allow the direct growth of adherent cells are, for example, mentioned in DE 197 53 598, DE 196 46 505, DE 44 17 079 and DE 196 46 505.
In these systems, a compatibility with the cell growth is achieved by operating with silicone-based sensors that are manufactured in thin-film technology. This technology, which has its own fields of application, e.g. the measurement on individual cells, causes some problems in the routine application in cell and tissue cultures. In particular, the silicone used is only a very special material for the adhesion of the cells. The silicone wafer that is moreover provided with further metallic materials in CMOS technology is a very special substrate for the adhesion of cells. As in the cell development it is precisely the interaction of the tissue cells with the substrate that plays a decisive role, a neutral substrate that is, above all, not influenced by electric currents (for amperometric sensors) and by diverse metal ions would be of utmost importance for the examination of more than just some specific questions.
If the behavior of cell cultures is detected on-line by means of electrochemical sensors, it cannot be avoided that a recalibration should be performed if the cells grow directly on the sensors. But this is not possible at present. Furthermore, it is just the manipulation at the cell or tissue culture that should be avoided during the on-line measurement. Normally, the arrays occupied by the cells can be used for maximally three days only. That is that the usability for the online measurement is limited to processes that are completely finished within this period or reach a point at which a significant statement about the course of the metabolism in question can be made.
Another problem will arise, if integrative total values are to be measured instead of punctual, local values of individual cells. Each of the sensors arranged one next to the other measures under another cell group and the given variability of biological systems complicates the correlation of the parameters measured in this way.
Finally, the production of sensors in thin-film technology implies efforts and expenses and therefore it is only economically practical for a larger volume.
However, the thick-film technology that offers higher flexibility and is much less expensive than the thin-film technology leads to the same disadvantages for the provided sensors as the thin-film technology.
Another problem is caused by the sterilization required for all parts that come into contact with the sample area. Here, a difference must be made between the simple disinfection that can be sufficient for short measuring phases and the sterilization that can be harmful for example for enzymes in biosensors. The most common sterilization method applied in laboratories is the high-pressure sterilization (in the autoclave).
Membranes are often used in combination with sensors. However, the membranes have been directly connected with the sensor so far. (For this, see WO 87/05624, DE 697 29 185 T1 and DE 199 24 856 A1, among others, where the functional selectivity is in the focus of attention for these sensors or the membrane referred to is an enzyme-carrying layer.)
The most important disadvantage of this technical solution is the fact that a multi-parametric analysis of the same sample and the recalibration required for the on-line measurement over longer periods are not possible.
In DE 694 11 732 T1 and EP 0289269, the specific function of the membrane is the exclusion of erythrocytes in blood analyses.
In WO 199 6032 64 A1, the cells are actually cultivated on a membrane, but the physiological parameters are measured by a sensor that is positioned in the cultivated tissue and thus influences the cultivation or changes the conditions.
The paper of D. Schepers, G. Schulze and W. Frenzel [“Spectrophotometric flow-through gas sensor for the determination of atmospheric nitrogen dioxide” (Analytika Chimica Acta 308 (1995) 109-114] describes a micro measuring cell for fluids that is particularly used for photometers or spectrometers, which preferably operate in a narrow band application, and contains two-dimensionally connected wafers that are provided with micro-channels in such a way that at least in one area two micro-channel sections are arranged parallel to each other and spatially separated from each other by a selective membrane chosen according to a substance to be analyzed so that an extraction path is formed, and the first wafer is provided with at least the first micro-channel section mentioned and its inlet and outlet openings or inlet and outlet channels combined with the ends of said channel section to transfer an analyte, and a second wafer is provided with at least the second micro-channel section mentioned and the inlet and outlet openings or inlet and outlet channel combined with the ends of said channel section to transfer an extraction means (E), and at least one wafer is transparent for a measurement light ray used for the measuring process or it is provided with a window range ensuring this, and the inlet and outlet of the measuring light ray is defined by the second micro-channel section used as an extraction channel so that, depending on the light source (L) used, an optical measuring path as long as possible is achieved.
The just mentioned micro measuring cell for fluids does not allow an on-line measurement of different physiological parameters of cells of one cell culture, in particular the measurement of soluble analytes and dissolved gases because specific sensors are not provided for the on-line measurement of soluble analytes and dissolved gases.
DE 198 60 547 A1 describes an affinity sensor for the detection of specific bond types consisting of a carrier substrate that is at least provided with two electrodes that are arranged at an equidistant distance one to the other and cover an area on both sides and at least said area is provided for the uptake of immobilized specific bonding partners that are capable to couple complementarily associated bonding partners directly or via further specific bonding molecules and said area is defined with a minimum width such that at least one complementarily associated bonding partner provided with an electrically conductive particle can be taken up in the mentioned area in such a manner that the possibility of the formation of a tunnel contact transfer is ensured between the particle and each electrode.
The just mentioned affinity sensor does not allow an on-line measurement on cells, in particular the measurement of soluble analytes and dissolved gases, because only specific couplings of bonding molecules can be electrically detected.